Chronic wounds that fail to respond to traditional therapeutic interventions afflict millions of people each year, and direct costs associated with treating these wounds are estimated at $5 to $10 billion annually. Eradication of pathogenic bacteria that have colonized chronic wounds is complicated by the propensity of these bacteria to form biofilms. A biofilm consists of a community of bacteria encompassed by an extracellular matrix that efficiently resists the action of antibiotics and the host immune response. Bacteria in the biofilm state are approximately 1000 times more resistant to antibiotics, and there are currently no reliable therapeutic strategies available for dispersing pre-formed biofilms. The co-founders of Agile Sciences have discovered a new class of small molecules, derived from a marine sponge natural product, that inhibits as well as disperses bacterial biofilms of both gram-positive and gram- negative bacteria, including bacteria commonly found in chronic wounds. These compounds are derived from a 2-aminoimidazole (2-AI) unit and represent the only class of non-toxic small molecules with the ability to disperse pre-formed bacterial biofilms. When applied to chronic wounds, these molecules are predicted to disperse bacterial biofilms into their more vulnerable planktonic state, so that the bacteria become significantly more susceptible to antibiotic therapies and to the host immune response. The goal of this work is to identify Agile molecules that are potent toward dispersing polymicrobial biofilms of chronic wound isolates under biomimetic conditions. Known structure-function relationships will guide the design of new analogues, which will be synthesized and screened for biofilm inhibition and dispersal of P. aeruginosa, S. aureus, and C. perfringens, bacteria that are most commonly isolated from chronic wounds. Once lead molecules are identified, their efficacy will be rigorously tested using in vitro wound biofilm models at the Center for Biofilm Engineering at Montana State University. These models include a drip-flow reactor, which will evaluate the ability of active analogues to remove biofilms of wound isolates under low-shear biofilm growth, as well as a biofilm and human cell co-culture scratch healing model, which will evaluate the synergistic relationship between active anti-biofilm molecules and antibiotics. The metric for success of the proposed work is to identify at least one analogue that disperses biofilms in all wound models, and demonstrates synergistic effects with antibiotics in the co-culture model by reducing scratch closure time. Analogues that successfully achieve these objectives will be advanced to a Phase II Study in which their therapeutic potential for promoting wound healing will be further assessed in vivo. PUBLIC HEALTH RELEVANCE: An estimated 1-2% of the population will suffer from persistent chronic wounds, and the difficulty in treating chronic wound infections has been attributed to bacteria9s ability to form biofilms. An innovative treatment for chronic wounds is being developed that removes bacteria from the biofilm state;this approach has the potential to significantly expedite the wound healing process.